Functionalized supports are useful in many applications, including: chromatography, solid-phase catalysts, solid-phase synthesis of polypeptides and oligonucleotides, and sequencing of polypeptides. The functionality of such supports can, in principle, be a property of the support itself, or more commonly, be the result of the modification of the surface of a substrate. The stability of surface modification is a critical factor whenever that route is chosen. Generally, if the modification is not stable, it means that the modifying reagent is released from the substrate thus contaminating the desired product. In chromatographic applications, this loss of surface modifying reagent is called "bleed". Most prior surface modifying reagents are not stable and their utility has been limited by that instability.
Silanes are the most commonly used surface modifying reagents. Snyder and Kirkland ("An Introduction to Modern Liquid Chromatography," Chapter 7, John Wiley and Sons, New York, 1979) disclose a representative chromatographic support with a surface monolayer of silane. This type of support is limited by the "bleed" of silane exposing the active silica substrate.
Chromatographic supports comprising polymeric silane phases on silica substrates are disclosed by Kirkland et al. (U.S. Pat. No. 3,722,181 and U.S. Pat. No. 3,795,313). These supports are produced using bi- or tri-functional silanes. The silane layers thus formed are relatively thick, limiting mass transfer with resulting poor chromatographic efficiency. Other polymeric silanes phases produced using dimethyl-1,1,4,4-tetramethoxydisilethylene and 1,1,3,3-tetramethyl-1,3-divinyldisilazane are disclosed by Jones et al. [J. Chromatogr., Volume 298, 389-397 (1984)]. The disilethylene reagent has two reactive sites on both Si atoms and can therefore form a polymeric phase with poor chromatographic efficiency. These phases are also limited by lack of control of the polymerization process. The disilazane reagent has only a single reactive site. Single point attachment of silanes to silica can lead to supports with limited stability.
Lork et al. [J. Chromatogr., Vol. 352, 199 (1986)] describe the use of bis-(n-octyldimethylsiloxane) in the preparation of siliceous chromatographic packings. Welsh et al. [J. Chromatogr., Volume 267, 39 (1983)] describe reversed-phase packings for HPLC produced by reacting silica with various disilazanes or disiloxanes. L. Boksanyi et al. [Advances in Colloid and Interface Science, Volume 6, 95 (1976)] used (CH.sub.3).sub.3 --Si--O--Si(CH.sub.3).sub.3 to prepare silane-modified silica surfaces. The disiloxanes employed in both references have three alkyl group substituents on each Si atom. These reagents bond to the surface only through a single Si atom. The disilazanes employed contain only a single reactive group again leading to single point attachment and thus limited stability.
Deschler et al. [Angew. Chem. Int. Ed. Engl., Vol. 25, 236 (1986)] discuss silanized siliceous surfaces produced with poly-condensed functionalized silanes from monomers of the type (RO).sub.3 Si--(CH.sub.2)--X--(CH.sub.2).sub.3 --Si(OR).sub.3. These reagents have three reactive groups on each Si atom and, therefore, like the disilethylene of Jones et al., can form polymeric phases with poor chromatographic efficiency.
Pines [EP 129074 A2, Dec. 27, 1984] describes an elastomeric finish to a substrate prepared by reacting a silane and a silanol to obtain a cross-linkable silicone intermediate which is thereafter reacted with a second silanol to obtain a silicone composition. This silicone composition, when catalyzed, can be used as an elastomeric finish on certain solid surfaces. These reagents are not suggested for use in chromatographic systems and since they are highly polymerized, their use in such applications is not advantageous as poor chromatographic efficiency is expected.
R. N. Lewis, U.S. Pat. No. 3,979,546, discloses surfaces that are rendered hydrophobic by the inorganic materials with alpha-alkoxy-omegasiloxanols. These reagents contain two different reactive groups which can react with each other to form undesirable polymeric structures.
Markiewicz et al. [Nucleic Acids Res., Spec. Publ., Volume 4, 185 (1978)] discuss 1,3-dihalo-1,1,3,3-tetraalkyldisiloxane as a silyl-protecting group for 3'--OH and 5'-OH groups of nucleosides. A review of similar reagents for use in protecting certain functional groups of soluble compounds in organic synthesis is given by M. Lalonde et al. [Synthesis, Volume 9, 817 (1985)]. Neither reference discloses using these reagents in any way other than as a protecting group during synthesis of soluble organic compounds.
Sindorf et al. [J. Amer. Chem. Soc., Volume 105, 3767 (1983)] discuss possible structures formed by reaction of bifunctional and trifunctional silanes with silica surfaces. They suggest, that in the presence of water, structures such as the following are formed. ##STR1## This structure is offered as only one of several present on the silica surface. There is no suggestion that this might be a preferred structure nor is any procedure disclosed for preparing silica substantially completely covered with a silane of such a structure.
There remains a need for highly stable supports for use in chromatography, polypeptide and oligonucleotide synthesis and polypeptide sequencing. These supports should offer a variety of functionalities for use in different applications, be stable during use, and be readily prepared.